Dynamics of the Myoglobin/Heme Complex: A Limited Proteolysis Perspective

Abstract

The dynamics and stabilities (i.e., physicochemical properties) of proteins and protein complexes are dictated by the solution environment. Changes in the solution environment may alter the distribution of states present in solution (i.e., the free-energy landscape) and thus their physicochemical properties. This work examines the effects of temperature (4 °C, 21 °C, and 37 °C), electrospray ionization buffers (ammonium acetate (AmAc) and triethylammonium acetate (TEAA)), and solvent (H2O and D2O) on the physicochemical characteristics of the myoglobin/heme complex using limited proteolysis (i.e., trypsin digestion) and ion mobility-mass spectrometry (IM-MS). Electrospray ionization mass spectra of peptides formed by limited proteolysis showed increased peptide abundances at higher temperatures and in the absence of buffer molecules relative to the intact precursors. Interestingly, peptide abundances in D2O are more consistent across the temperature range studied relative to the peptide abundances in H2O, which demonstrates that D2O not only stabilizes proteins but acts as a kinetic trap for specific protein conformations. As an example, buffer components and D2O stabilize the myoglobin/heme interaction, as is evidenced by the modulation of peptide 64–77, an α-helical region that interacts with heme, and by shifts in the abundances of unfolded protein precursors. Lastly, IM-MS analysis of digested peptides suggests that AmAc alters the tertiary structure of the myoglobin/heme complex, whereas TEAA alters the secondary structure of the complex. The preservation of secondary structure in TEAA is further illustrated by the shift in arrival time distributions of 1+ ions in TEAA relative to the 2+, 3+, 4+, and 5+ ions present in other buffer conditions. Overall, the data show that buffer-solvent interactions govern the hydration of myoglobin and consequently, the physicochemical properties of the complex.